Abstract HIV-1TatactivatesviraltranscriptionandlimitedTat-transactivationcorrelateswithlatencyestablishment.We postulateda?block-and-lock?functionalcureapproachbasedonpropertiesoftheTat-inhibitordidehydro- Cortistatin A (dCA). HIV-1 transcriptional inhibitors could block ongoing viremia during antiretroviral therapy (ART), locking the HIV promoter in persistent latency. We investigated this hypothesis in human CD4 +T cells isolated from aviremic individuals. CombiningdCA withARTaccelerates HIV-1 suppression and prevents viral rebound after treatment interruption, even during strong cellular activation. We show that dCA mediates epigenetic silencing by increased nucleosomal occupancy at Nucleosome-1, restricting RNAPII recruitment to the HIV-1 promoter. The efficacy of dCA was studied in the bone marrow-liver-thymus (BLT) mouse modelof HIVlatencyandpersistence.AddingdCAtoARTsuppressedmicesystemicallyreducesviralmRNAintissues. Moreover,dCAsignificantlydelaysandreducesviralreboundlevelsupontreatmentinterruption.Altogetherthis workdemonstratesthepotentialof?block-and-lock?curestrategies. ATatinhibitorisunlikeanyotherHIVinhibitor,asdurationoftreatmentimpactstheoutcome,becauseof the feedback nature of the Tat-TAR activity and because epigenetic marks deposited at the HIV-1 promoter accrue over time. We hypothesized that over time transcriptional repression could be pushed past a certain threshold where viral reactivation from latency isextremelydifficult toovercome,blocking-and-locking HIV into sustained latency. The additive activity of dCA also supports the notion that adding Tat inhibitors to front-line treatment might lead to faster suppression and potentially reduce the size of the established reservoir. It is emergingfromstudiesofindividualsonveryearlyARTtreatmentthatasmallerreservoirsizedirectlytranslates intobetterviralcontrol(14). ThegeneticbarriertoviralresistancetodCAinvitrowasinvestigated,andunexpectedlybutnottoosurprising, mutations in Tat and TAR were not found, since these are extremely conserved. Instead, viruses resistant to dCA developed very high Tat-independent basal transcription. We identified a combination of mutation in the LTRpromoterthatincreasedbasaltranscriptionalactivity,andmodificationsinNefandVprthatincreasedNF- k?Bactivity.Wehypothesizethesevirusesmaynotdevelopinvivo,wereasontheirincreasedtranscriptionfitness andinabilitytocontrolentryintolatencymayultimatelybedetrimental,leadingtohighcytopathiceffectsand/or clearancebytheimmunesystem. Looking ahead we have three main goals: 1) using BLT mice to understand the relationship between dCA treatmenttimeandreductionsinresidualviralRNAproductionandhowthattranslatesindelayingviralrebound aftertreatmentinterruption;?2)studytheimpactofdCAasfront-linetherapyonthesizeoftheestablishedviral reservoirduringacutephasetreatment;?and3)studymechanismsofviralresistancetodCAinvivo.